ABSTRACT Tuberculosis (TB) remains a major public health problem in developing countries. More recently, the prevalence of non-tuberculous mycobacteria (NTM) has also been increasing worldwide in both developed and developing countries. NTM infection is clinically undistingable from tuberculosis and poses significant challenges in patient management, especialy in patients with chronic pulmonary TB (known to have 18% of NTM infections). The sputum smear, the most widely used diagnostic tool for TB is more than 100 years old, misses to detect half of tuberculosis cases, and is unable to differentiate between mycobacterial species. The World Health Organization (WHO) recommends Xpert MTB/RIF assay and other molecular tools, however, Xpert MTB/RIF is less sensitive than sputum culture (the current gold standard) and does not address the differential with growing NTM infections. In this application, we propose to leverage the scientific infrastructure and collaboration between the Center for Innovation in Global Health Technologies (CIGHT) at Northwestern University and the SEREFO HIV/TB Laboratory in Mali, at the University of Bamako (USTTB), to evaluate a new highly sensitive Multiplex MTB/NTM assay that can differentiate, at least M. tuberculosis complex (MTBC, TB causative agent) from the treatable and the most common NTM, M. avium complex (MAC). Preliminary data from the new assay suggest a performance superior to Xpert TB/RIF and comparable to sputum culture. The specific aims of this project are to evaluate the assay on fresh sputum samples from 1) patients with chronic TB-like disease, and 2) TB suspect patients with negative sputum smear. The study will evaluate the assay sensitivity, specificity, positive and negative predictive values for both MTBC and NTM. The development and validation of such tools will significantly contribute into the fight against both TB and NTM diseases worldwide. Finally, the project assembled an excellent and complementary team to ensure the success of this multidisciplinary and highly innovative project.